We report the development of reversibly attached phospholipid bilayer (PLB)-functionalized membrane pores that enabled reusability of the membrane matrix as well as the phospholipid. The functionalized architecture was constructed based on electrostatic interactions, which facilitate the reversible attachment detachment sequence of the functional moieties within membrane pores. To demonstrate potential application, an enzyme, glucose oxidase (GOx), was electrostatically immobilized within the PLBfunctionalized membrane and enzymatic catalysis was conducted membrane demonstrated satisfactory activity and stability. Convective flow of the substrate solution resulted in significantly higher activity than diffusive flow. Then, the enzyme was detached keeping the functional PLB backbone intact. Detachment of the enzyme without affecting the functional activity of PLB backbone permits attachment of fresh enzyme. In addition, reusability of the phospholipids is also of great importance as they have wide range of applications, but their usage is limited by higher cost. We have demonstrated the detachment of the PLB from the membrane using a simple technique. Characterization of the detached phospholipid confirmed retention of the original structural and functional properties as exhibited before attachment. To the best of our knowledge, this is the first study on reversible PLB formation within membrane pores and demonstration of a detachment technique, while maintaining the structural and functional properties of the phospholipid.